Search

Contains fulltext : 49601.pdf (Publisher’s version ) (Open Access), Heparan sulfate (HS) proteoglycans are major anionic glycoconjugates of the glomerular basement membrane and are thought to contribute to the permeability properties of the glomerular capillary wall. In this study we evaluated whether the development of (micro) albuminuria in early human and experimental diabetic nephropathy is related to changes in glomerular HS expression or structure. Using a panel of recently characterized antibodies, glomerular HS expression was studied in kidney biopsies of type I diabetic patients with microalbuminuria or early albuminuria and in rat renal tissue after 5 months diabetes duration. Glomerular staining, however, revealed no differences between control and diabetic specimens. A significant (p < 0.05) approximately 60% increase was found in HS N-deacetylase activity, a key enzyme in HS sulfation reactions, in diabetic glomeruli. Structural analysis of glomerular HS after in vivo and in vitro radiolabeling techniques revealed no changes in HS N-sulfation or charge density. Also HS chain length, protein binding properties, as well as disaccharide composition did not differ between control and diabetic glomerular HS samples. These results indicate that in experimental and early human diabetic nephropathy, increased urinary albumin excretion is not caused by loss of glomerular HS expression or sulfation and suggest other mechanisms to be responsible for increased glomerular albumin permeability.

Item does not contain fulltext, The biosynthesis of heparan sulfate, present on the cell surface and in the basal lamina surrounding cells, is a multistep process in which each step is mediated by a specific enzyme. The initial modification of the precursor polysaccharide, N-deacetylation followed by N-sulfation of selected N-acetyl-D-glucosamine residues, is catalyzed by the enzyme glucosaminyl N-deacetylase/N-sulfotransferase (NDST). This event is a key step that regulates the overall sulfate content of the polysaccharide. Here, we report on the effects of NDST deficiency on Ca2+ kinetics in myotubes from NDST-1- and NDST-2-deficient mice, indicating a novel role for heparan sulfate in skeletal muscle physiology. Immunostaining for specific heparan sulfate epitopes showed major changes in the heparan sulfate composition in skeletal muscle tissue derived from NDST-1-/- mice and NDST-/- cultured myotubes. Biochemical analysis indicates a relative decrease in both N-sulfation and 2-O-sulfation of skeletal muscle heparan sulfate. The core protein of heparan sulfate proteoglycan perlecan was not affected, as judged by immunohistochemistry. Also, acetylcholine receptor clustering and the occurrence of other ion channels involved in excitation-contraction coupling were not altered. In NDST-2-/- mice and heterozygous mice no changes in heparan sulfate composition were observed. Using high-speed UV confocal laser scanning microscopy, aberrant Ca2+ kinetics were observed in NDST-1-/- myotubes, but not in NDST-2-/- or heterozygous myotubes. Electrically induced Ca2+ spikes had significantly lower amplitudes, and a reduced removal rate of cytosolic Ca2+, indicating the importance of heparan sulfate in muscle Ca2+ kinetics.

Contains fulltext : 184929.pdf (publisher's version ) (Closed access), A new assay was developed to measure the N-deacetylase activity of the glucosaminyl N-deacetylase/N-sulfotransferases (NDSTs), which are key enzymes in sulfation of heparan sulfate (HS)/heparin. The assay is based on the recognition of NDST-generated N-unsubstituted glucosamine units in Escherichia coli K5 capsular polysaccharide or in HSs by monoclonal antibody JM-403. Substrate specificity and potential product inhibition of the NDST isoforms 1 and 2 were analyzed by comparing lysates of human 293 kidney cells stably transfected with mouse NDST-1 or -2. We found HSs to be excellent substrates for both NDST enzymes. Both NDST-1 and -2 N-deacetylate heparan sulfate from human aorta ( approximately 0.6 sulfate groups/disaccharide) with comparable high efficiency, apparent Km values of 0.35 and 0.76 microM (calculation based on [HexA]) being lower (representing a higher affinity) than those for K5 polysaccharide (13.3 and 4.7 microM, respectively). Comparison of various HS preparations and the unsulfated K5 polysaccharide as substrates indicate that both NDST-1 and -2 can differentially N-sulfate polysaccharides already modified to some extent by various other enzymes involved in HS/heparin synthesis. Both enzymes were equally inhibited by N-sulfated sequences (>or=6 sugar residues) present in N-sulfated K5, N-deacetylated N-resulfated HS, and heparin. Our primary findings were confirmed in the conventional N-deacetylase assay measuring the release of 3H-acetate of radiolabeled K5 or HS as substrates. We furthermore showed that NDST N-deacetylase activity in crude cell/tissue lysates can be partially blocked by endogenous HS/heparin. We speculate that in HS biosynthesis, some NDST variants initiate HS modification/sulfation reactions, whereas other (or the same) NDST isoforms later on fill in or extend already modified HS sequences.

Item does not contain fulltext

Proteoglycans (PGs), biosynthetically labelled with [S-35]sulphate, were isolated from mouse mastocytoma tissue. Chromatography on antithrombin (AT)-Sepharose resulted in the separation of the S-35-labelled PGs into three fractions: PGs with no affinity for the gel (NA-PGs), PGs with low affinity (LA-PGs), and PGs with high affinity (HA-PGs) for antithrombin. Whereas NA-PGs contained almost exclusively chondroitin sulphate (CS), the AT-binding PGs contained 80-85% heparin and 15-20% CS. [S-35]CS-containing macromolecules obtained from the HA-PG fraction after removal of the heparin polysaccharide chains were rechromatographed on AT-Sepharose. A majority of these S-35-labelled macromolecules no longer showed affinity for AT. These experiments indicate that the [S-35]CS recovered in the AT-binding PGs is present in hybrid PGs. Polysaccharide chain-length determination demonstrated that the heparin chains were somewhat larger (M(r) similar to 30000) than the CS chains in the NA-PGs (M(r) similar to 25000). CS chains in the hybrid PGs were slightly smaller (M(r) similar to 20000). Characterization of the sulphated CS disaccharides from NA- and HA-PGs showed that they contained similar amounts (20 %) of disulphated disaccharides of [GlcA-GalNAc(4,6-di-OSO3)] type. The monosulphated CS-disaccharides were O-sulphated at C-4 of the galactosamine units. Analysis by gel chromatography of the [S-35]CS components isolated from HA-PGs after heparinase treatment showed that a major portion of these contained one CS chain only. Calculations of the number of CS and heparin chains in AT-binding PGs, based on polysaccharide composition and polysaccharide chain length, indicate that all heparin-containing PGs are hybrids.

Contains fulltext : 216430.pdf (Publisher’s version ) (Open Access)